Neuropeptides and other peptide hormones are synthesized as precursor proteins that are then cleaved by limited proteolysis. Recent findings indicate that the processing of prohormones may be due to the action of a newly described family of prohormone convertases (PCs) that are evolutionarily related to bacterial subtilisin. The mode of action, localization, and tissue specificity of these proteases are under intense investigation. The known convertases, as well as prohormone processing in general, are remarkably conserved from yeast to mammals. Consequently, the yeast and Aplysia invertebrate models have contributed significantly to our overall understanding of prohormone processing. However, the story is far from complete; further development and study of appropriate model hormone systems are necessary to answer critical questions. Although heterologous co-transfection studies have indicated that the convertases can process a variety of prohormones, do these convertases in fact carry out such hydrolysis in vivo? What is their mechanism of action? Does processing result from the consecutive action of more than one enzyme? Research described for the next funding period will continue to exploit a prohormone processing model in Aplysia californica involving egg-laying hormone (ELH), which is synthesized as a precursor by bag cell neurons. Related genes are also expressed in the atrial gland, an exocrine organ. Our findings, as well as reports of others, have demonstrated that the atrial gland and the bag cell neurons express a variety of convertases (including PC1, PC2, furin1, and furin2) that are candidate processing enzymes of the ELH-like precursors. Thus, the Aplysia model uniquely allows comparison of the processing of homologous gene products in two different tissues, neuroendocrine (bag cells) and exocrine (atrial gland), at two levels of complexity by essentially identical convertases. Research in the next project period will further define the role of the prohormone convertases in the processing of ELH-like precursors. Overexpression procedures will be developed, using bacterial and eucaryotic systems, to make available sufficient amounts of ELH-like precursors and prohormone convertases for biochemical and enzymic studies. Biochemical studies investigating the kinetics of convertase-mediated prohormone processing will provide answers for the questions raised above. Site-directed mutagenesis will probe those structural features of the precursors that are-critical for processing. In addition, we propose to investigate major regulatory molecules that impact on prohormone convertase biosynthesis, activation, stability, and activity. In the longer term, these investigations will substantially develop the Aplysia model of prohormone processing and provide a solid foundation for future studies. Better understanding of prohormone processing should provide important insights into disease processes involving idiopathic hormone hypofunction.